National Heart and Lung Institute, Imperial College London, London, United Kingdom.
Host Defence Unit, Royal Brompton Hospital, London, United Kingdom.
PLoS One. 2018 Dec 11;13(12):e0208018. doi: 10.1371/journal.pone.0208018. eCollection 2018.
The diagnosis of pulmonary non-tuberculous mycobacterial disease (pNTM) is dependent on the isolation of NTM in culture, which is prone to overgrowth and contamination and may not capture the diversity of mycobacteria present, including rare or unidentified species. This study aimed to develop a culture independent method of detecting and identifying mycobacteria from sputum samples using partial sequencing of the hsp65 gene. DNA was extracted from sputum samples from subjects with pNTM and disease controls. Multiplexed partial sequencing of the hsp65 gene was performed using the Illumina MiSeq and custom primers. A reference database of hsp65 sequences was created for taxonomy assignment. Sequencing results were obtained from 42 subjects (31 cases, 11 controls). Mycobacterial sequences were identified in all subjects. In 90.5% of samples more than one species was found (median 5.5). The species isolated in culture was detected by sequencing in 81% of subjects and was the most abundant species in 62%. The sequencing of NTM from clinical samples reveals a far greater diversity than conventional culture and suggests NTM are present as communities rather than a single species. NTM were found to be present even in the absence of isolation in culture or clinical disease.
肺部非结核分枝杆菌病（pNTM）的诊断依赖于分枝杆菌的培养分离，而培养容易出现过度生长和污染，并且可能无法捕捉到存在的分枝杆菌的多样性，包括罕见或未鉴定的物种。本研究旨在开发一种使用 hsp65 基因部分测序从痰样本中检测和鉴定分枝杆菌的非培养依赖方法。从 pNTM 患者和疾病对照者的痰样本中提取 DNA。使用 Illumina MiSeq 和定制引物对 hsp65 基因进行多重部分测序。创建了 hsp65 序列的参考数据库以进行分类学分配。从 42 名受试者（31 例，11 例对照）中获得了测序结果。在所有受试者中均鉴定出分枝杆菌序列。在 90.5％的样本中发现了一种以上的物种（中位数为 5.5）。在 81％的受试者中通过测序检测到培养分离的物种，在 62％的受试者中该物种是最丰富的。从临床样本中对 NTM 的测序揭示了比传统培养更广泛的多样性，并表明 NTM 以群落而不是单一物种存在。即使在培养分离或临床疾病不存在的情况下也发现存在 NTM。
